Pharmacological and SAR analysis of the LINS01 compounds at the human histamine H1, H2, and H3 receptors

Article abstract of DOI:10.1111/cbdd.13387

Histamine is a transmitter that activates the four receptors H₁R to H₄R. The H₃R is found in the nervous system as an autoreceptor and heteroreceptor, and controls the release of neurotransmitters, making it a potential drug target for neuropsychiatric conditions. We have previously reported that the 1-(2,3-dihydro-1-benzofuran-2-yl)methylpiperazines (LINS01 compounds) have the selectivity for the H₃R over the H₄R. Here, we describe their pharmacological properties at the human H₁R and H₂R in parallel with the H₃R, thus providing a full analysis of these compounds as histamine receptor ligands through reporter gene assays. Eight of the nine LINS01 compounds inhibited H₃R-induced histamine responses, but no inhibition of H₂R-induced responses was seen. Three compounds were weakly able to inhibit H₁R-induced responses. No agonist responses were seen to any of the compounds at any receptor. SAR analysis shows that the N-methyl group improves H₃R affinity while the N-phenyl group is detrimental. The methoxy derivative, LINS01009, had the highest affinity.

Full text of DOI:10.1111/cbdd.13387

DR JOÃO PAULO SANTOS FERNANDES (Orcid ID : 0000-0002-9089-273X)
Article type : Research Letter
Pharmacological and SAR analysis of the LINS01 compounds at the human
histamine H1, H2 and H3 receptors
Michelle Fidelis CORRÊAa, Álefe Jhonatas Ramos BARBOSAa, Gustavo Ariel
Borges FERNANDESa, Jillian G. BAKERb and João Paulo dos Santos
FERNANDESa,
aDepartamento de Ciências Farmacêuticas, Universidade Federal de São Paulo, Rua São
Nicolau 210, Diadema-SP 09913-030, Brazil
bCell Signalling Research Group, School of Life Sciences, University of Nottingham,
Nottingham NG7 2UH, United Kingdom
Keywords: antihistamines, histamine receptor ligands, H3R antagonists, ligand
efficiency analysis, structure-activity relationships.
Short Running Title: Pharmacology of LINS series at histamine receptors
Abstract
Histamine is a transmitter that activates the four receptors H1R to H4R. The H3R is
found in the nervous system as an auto and heteroreceptor, and controls the release
of neurotransmitters, making it a potential drug target for neuropsychiatric conditions.
We have
previously reported
that the 1-(2,3-dihydro-1-benzofuran-2-
yl)methylpiperazines (LINS01 compounds) have selectivity for the H3R over the H4R.
Here we describe their pharmacological properties at the human H1R, H2R in parallel
Corresponding author. Tel.: +55-11-4044-0500; e-mail: joao.fernandes@unifesp.br
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13387
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with the H3R, thus providing a full analysis of these compounds as histamine
receptor ligands through reporter gene assays. Eight of the nine LINS01 compounds
inhibited H3R-induced histamine responses but no inhibition of H2R-induced
responses were seen. Three compounds were weakly able to inhibit H1R-induced
responses. No agonist responses were seen to any of the compounds at any
receptor. SAR analysis shows that the N-methyl group improves H3R affinity whilst
the N-phenyl group is detrimental. The methoxy derivative, LINS01009, had the
highest affinity.
Histamine is a biogenic amine that has important clinical and pathophysiological
roles in allergy, gastric acid secretion and inflammatory diseases. Histamine effects
occur by activation of histamine receptors of which there are four subtypes and all
are G-protein coupled receptors (GPCRs), namely H1R, H2R, H3R and H4R.[1,2]
The H1R is a Gq-coupled receptor and activation of this is important in allergy. There
are many H1R-antagonists in widespread clinical use for allergies such as hayfever
(including oral agents e.g. certirizine, loratadine and chlorpheniramine, and topical
preparations e.g. mepyramine). The H2R is a Gs-coupled receptor. Activation is
important in the production of gastric acid and H2R-antagonists are widely used to
reduce gastric acid production (e.g. ranitidine).[1] The H3R is a Gi-coupled receptor
and present in the CNS and on peripheral neurons. It is important for inhibiting the
release of histamine (as an autoreceptor) but also negatively regulating the synthesis
and release of many neurotransmitters. H3R-antagonists have been investigated as
potential treatments for several CNS disorders, including sleep disorders,
schizophrenia, epilepsy, attention deficit and hyperactivity disorder and even obesity.
There is also potential for H3R-antagonists to have effects in neurodegenerative
disorders such as Alzheimer’s, Parkinson’s disease and Huntington’s chorea.[3-6]
Recently, a H3R antagonist (pitolisant) has been approval for the maintenance of
wakefulness in patients with narcolepsy.[7,8] The H4R is also a Gi-coupled receptor
and is most widely expressed in bone marrow and on white blood cells and shown to
be an important target to treat inflammation,[2] although to date no H4R antagonist is
available for clinical use. There is a considerable overlap between the selectivity of
ligands for the H3R and H4R, with many H3R ligands having high affinity for the H4R,
however some H4R selective ligands have been identified (e.g. JNJ-7777120).[1,2] We
have recently developed a series of compounds, originally designed based on the
H4R selective antagonist JNJ-7777120 chemophore, the 1-(2,3-dihydro-1-
benzofuran-2-yl)methylpiperazines (LINS01) series (Figure 1).
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Initial studies examining [3H]-histamine binding to H3R and H4R membrane
preparations of the first four compounds suggested micromolar affinities for these
compounds, with higher affinity for the H3R than H4R.[9] A further study of other
LINS01 compounds in HEK293 cells transiently expressing the H3R or H4R
suggested the LINS01 compounds are antagonists or weak inverse agonists but
once again that they had higher affinity for the H3R than the H4R.[10] Given the
surprising finding that these LINS01 compounds had higher affinity for H3R than H4R,
we decided to investigate the pharmacological profile of these ligands at the other
histamine receptors, the H1R and H2R.
The LINS01 compounds (1a-1i) were synthesized as previously reported by our
group (Figure 2, see supporting Information).[9-11] In case of the non-substituted
phenols (to obtain compounds 1a-d), the commercial 2-allylphenol was used as
starting material. In case of R’-substituted molecules, the corresponding phenols 2e-
h were used as starting material, which were allylated with allyl bromide in
conventional or ultrasonic conditions, with good to excellent yields. The yields in
conventional or ultrasonic methods are comparable, however the ultrasonic
methodology saved reaction time and thus was considered advantageous. The allyl-
phenyl ethers 3e-h were then thermally isomerized (>200 °C) to access the 2-
allylphenols (4a-h) with good yield using a microwave-assisted Claisen
rearrangement approach, using DMF as solvent. The dihydrobenzofurans (5a-h)
were obtained through iodine-promoted cyclization from the corresponding ortho-
allylphenols in an eco-friendly medium, using water or a mixture of ethanol-water as
solvent. Finally, the iodinated heterocycle was used to alkylate the N-substituted
piperazines in aprotic solvent (THF), with potassium carbonate as base, with
moderate yields. A novel microwave-assisted methodology was employed to avoid
the considerable excess of 1-phenylpiperazine to obtain compounds 1d and 1i,
leading to moderate to good yields (50-70%). Although this method gave comparable
yields to the conventional methodology, it also saved reaction time and required less
1-phenylpiperazine (1.1 eq.) indeed, then comprising the green chemistry
principles.[12] The spectroscopic data for the final compounds (see Supporting
Information) and intermediates are in accordance to the literature reports.[9-11] None
of the LINS01 molecules were previously evaluated in H1R and H2R, and the
compound 1i was not evaluated in H3R before. This is also the first report on the
affinity data for these compounds through reporter gene assays.
Histamine stimulated a response that was 2.3 ± 0.1 fold over basal in the H1-SRE-
luciferase cells (pEC50 6.78 ± 0.04; n=8, Figure 3a,b). This response was readily
inhibited by mepyramine, a histamine H1R antagonist, causing a parallel rightward
shift of the histamine concentration response and yielding a pKD value of 8.53 ± 0.05
(n=9). ICI162846 (an H2R antagonist) and JNJ-7777120 (an H4R antagonist) were
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not able to antagonize the histamine response at concentrations of up to 10 µM.
Clobenproprit (an H3R/H4R antagonist) caused a small rightward shift of the
histamine response (pKD 5.01 ± 0.11; n=9). Thus histamine responses are readily
antagonized by mepyramine, but not ICI182846, clobenpropit or JNJ-7777120,
confirming the presence of the H1R in this cell line.[13]
Histamine also stimulated a response (pEC50 7.50 ± 0.03; n=8) in the H2-CRE-SPAP
cells that was 3.8 ± 0.1 fold over basal (Figure 3c,d). This response was inhibited by
the H2R antagonist ICI162846 (pKD 8.71 ± 0.04; n=9) whereas neither mepyramine
nor clobenpropit nor JNJ-7777120 were able to cause a rightward shift of the
histamine concentration response curve. This confirms the presence of the H2R in
this cell line.[13]
Histamine did not stimulate a response, nor inhibit the basal response, on its own in
the CHO-H3-CRE-SPAP cells, indicating the absence of a Gs-coupled stimulatory
histamine receptor.[14] Histamine did however inhibit forskolin-induced CRE-SPAP
production (pEC50 6.66 ± 0.04; n=9) in a manner consistent with stimulation of a Gi-
coupled histamine receptor (Figure 3e,f). This histamine Gi-induced agonist
response was inhibited by clobenproprit (pKD 9.18 ± 0.06; n=9). As expected, neither
mepyramine nor ICI162846 were able to inhibit the histamine responses in this cell
line. JNJ-7777120 cause a small rightward shift of the histamine-concentration
response curve (pKD 5.45 ± 0.07; n=4), very similar to that previously reported for the
human H3R (pKD 5.29) and very different from that for the histamine H4R (pKD
8.40).[15] Thus the presence of the histamine H3R was confirmed in this cell line.[14]
None of the LINS01 compound series stimulated any agonist responses in any of the
cell lines (Figure 4). With the exception of 1a, the LINS01 compounds were able to
inhibit the H3R histamine induced response to yield measureable pKD values. The
compound 1g had the highest affinity (pKD 7.18) followed by 1h (pKD 6.99; Figure 4,
Table 1). Increasing concentrations of 1g caused progressive rightward shifts of the
histamine concentration response curve in keeping with competitive antagonism at
the H3R (Figure 4e). Three compounds did cause a small rightward shift of the
histamine response in CHO-H1-SRE-luciferase cells giving measureable pKD values.
Interestingly, 1a, that did not have any measureable affinity for the H3R, had a very
weak affinity for the H1R. None of the LINS01 ligands had any measureable affinity
for the H2R.
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As the LINS01 molecules were originally designed to be similar to the H4R selective
antagonist JNJ-7777120,[9] it was expected that these molecules would also be H4-
selective. Indeed, two molecules (LINS01005 – 1d and LINS01007 – 1e) appear to
have a dose-dependent anti-inflammatory effect in a murine mouse model of asthma
that correlated with the H4R affinity.[9,10] However, these studies have also shown
that most of LINS01 molecules actually have higher affinity for the H3R than the
H4R.[9,10] It was therefore important to know the pharmacological profile of these
compounds for the human H1R and H2R.
The LINS01 compounds, together with mepyramine (selective H1R antagonist), ICI
162864 (selective H2R antagonist), clobenpropit (H3R/H4R antagonist) and JNJ-
7777120 (H4R antagonist) were studied using the functional read out of reporter
gene assays in CHO cell lines stable expressing each of the human H1R, H2R and
H3R. For the Gq-coupled H1R, an SRE-reporter was used, whereas for the Gs-
coupled H2R and Gi-coupled H3R (both of which affect cAMP) a CRE-reporter was
used.[13,14] Reporter gene assays have several advantages. They can be used to
examine responses from receptors without the need for large extracellular or
intracellular tags, which have the potential to alter the ligand-receptor binding or
receptor-effector coupling respectively. The longer agonist incubation times (here, 5
h at 37 °C) means that the interaction between the ligand and the receptor is highly
likely to reach equilibrium allowing the use of pharmacological analyses such as the
rightward shift of agonist concentration responses (Gaddum equation)[13,14] to assess
ligand affinity (often not possible with very short term second messenger responses
such as calcium). Thus the efficacy of ligands (ability to stimulate a response) and
the affinity of antagonist ligands can be assessed within one assay. Also unlike
second messenger assays (e.g. calcium, cAMP), being downstream responses,
reporter gene assays also read out from several different signaling cascades (e.g.
cAMP and MAP kinase).[16]
None of the LINS01 compounds stimulated any agonist responses at any of the
histamine receptors studied. Although each of the selective antagonists inhibited
histamine responses with high affinity at their respective receptors, no inhibition of
histamine responses were seen with the LINS01 compounds in the H2R expressing
cells. There is therefore no evidence for any ligand-receptor interaction for the
LINS01 compounds with the H2R. Three of the LINS compounds had a measureable
affinity, albeit low, for the human H1R. Of these, 1a is most interesting in that this
compound was the one compound from the LINS01 series that had no affinity for the
H3R. The compound 1a therefore appears to have H1R selectivity (although the H1R
affinity is very poor). All compounds with the exception of 1a antagonized histamine
responses yielding measurable pKD values.
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Looking into the SAR on the H3R affinity, the importance of the N-alkyl group (R) is
clear. The unsubstituted compound (1a) had no affinity for the H3R although did for
the H1R. Substitution with either N-methyl (1b) or N-allyl (1c) groups led to improved
H3R affinity, suggesting there is an additional hydrophobic interaction of the R-group
in the binding site. The N-phenyl group (1d) also decreased H3R affinity, possibly
due steric hindrance or by decreasing the basicity of the nitrogen. Thus the N-alkyl
group is important for H3R affinity, and possible lack of it important for H1R affinity.
Several H3R ligands reported in the literature present the N-arylpiperazine moiety
such as DL-80[17] which present medium to low affinity to H3R, but with certain
selectivity over H4R. However these compounds present a longer linker than the
LINS01 compounds, and thus the position of the most basic nitrogen changes
accordingly. Docking studies[18,19] suggest that a salt bridge between a basic nitrogen
and Glu206 is a key interaction to the antagonist activity. Figure 5 shows the
proposed interactions of several antagonists with H3R, and the hypothetical
interaction of 1g. The alignment of UCL2190, DL-80 and 1g (Figure 5) suggests that
the nitrogens supposed to interact with Glu206 and the aromatic rings of these
molecules can occupy the same positions in space, supporting this hypothesis.
We have previously shown that only compounds 1e and 1f showed any measureable
affinity for the H4R, suggesting that the R’ group may play the role in the H4R affinity.
Compound 1e also presented similar pKi for both receptors (~6.0).[10] The high
homology between these two receptors explains the non-selectivity of several
ligands from literature. It is known that H3R and H4R share two highly conserved
amino acids in the binding site, an aspartate (Asp114 and Asp94, respectively) and
an glutamate (Glu206 and Glu182, respectively), which interact with the primary
amine and imidazole, respectively, of histamine. This explains the higher affinity of
histamine for these receptors than for H1R and H2R.[20] The interaction with Glu206
generally determines the affinity of H3R ligands and possibly the low affinity of these
ligands to H1R and H2R, since Glu206 is not present in these receptors. By
observing the present results, we believe that the more basic nitrogen of LINS01
compounds interact with Glu206 by analogy.
The results also suggest that some volume in the R’ region is tolerated by H3R. As
can be noted in Table 1, compounds 1e-1h present a substituent in R’ and the
binding affinities kept similar. The presence of the bulky t-butyl group in 1h
maintained the binding affinity value ~7.0, corroborating this hypothesis. However,
the more polar methoxy group led to better affinity (pKD 7.18), suggesting that polar
atoms can improve the potency without increasing lipophilicity indeed. Molecules
with excessive lipophilicity can lead to pharmacokinetic issues in the development
stage, and thus should be avoided at initial stages.[21]
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Ligand efficiency metric analysis (see Supporting Information) is a suitable approach
to evaluate the contribution of each atom of a given molecule to the binding
affinity.[21,22] The concept of ligand efficiency (LE) arouse from the need to measure
whether a potency of a ligand derives from an adequate fit with the target or simply
by doing many nonspecific interactions with it.[23] Literature considers that a good LE
value to a lead-like molecule is at least >0.3, while market drugs present an average
LE value >0.45.[21,22] All LINS01 compounds presented LE >0.3 regarding H3R
affinity, and thus can be considered promising compounds for further development.
Compounds 1b, 1f and 1g must be highlighted due to their high LE values (>0.52),
and with exception of the N-phenyl derivatives 1d and 1i, the LINS01 molecules
present LE values comparable to drugs that are doing well in the market. The fit
quality (FQ) is a size-independent measurement of LE, i.e. it allows direct
comparison of the values independently from the molecular size.[24] Again, the
LINS01 compounds present very good values for FQ and are considered interesting
lead compounds.
Derivations of LE concept considering the lipophilicity also appeared, such as the
lipophilicity-corrected ligand efficiency (LELP) and the lipophilic ligand efficiency
(LLE), in order to ponder the LE with the lipophilicity (mainly measured by clogP, see
Supporting Information).[25,26] Excessive hydrophobicity should be avoided during
drug discovery process because it limits the water solubility (which directly impacts
on pharmacokinetics) and favors nonspecific interactions with biological proteins.
The optimum LELP values are considered in the range from -10 to +10, while LLE
values should be between 5 and 7.[21] Once again, the N-phenyl derivatives 1d and
1i showed LELP values outside the desired values, indicating that the phenyl group
led to excessive lipophilicity. This lipophilicity can be verified by the high clogP
values for these molecules. Considering that the lipophilicity usually increases during
the development,[25] these molecules are not promising for further modifications, or
these modifications should consider more hydrophilic groups. The LLE value of 1g is
within the optimal value, indicating that the insertion of methoxy group in R’ led to
improved affinity and contributed to reduce the lipophilicity of the molecule at the
same time, leading to a clogP value similar to that from the non-substituted molecule
1b.
Group efficiency (GE) is also a metric value to evaluate the contribution of an
inserted group to the potency.[27] LINS01 molecules present a common
dihydrobenzofuranyl-methylpiperazine core with structural modifications in the basic
nitrogen (R) and in the aromatic ring (R’). As can be noted in Tables 1 and 2,
modifications in the R-group lead to important variations in the binding affinities. The
presence of N-methyl group led to important improvement in the affinity to H3R (GE =
2.29), indicating that this group should be maintained to good H3R affinity. The N-
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allyl group also increased the affinity, but as it present 3 heavy atoms its efficiency
was lower than N-methyl (GE = 0.85). In counterpart the N-phenyl group of 1d
presented low GE value. The higher the GE value, the higher the contribution of the
group to increase the potency. For example, the addition of a group with one heavy
atom (HA = 1) and a GE value of 0.52 in a molecule with molecular weight <300
means an increase of 2.3-fold in potency.[21,27] This means that the N-methyl group
contributes very importantly to the binding affinity, while the N-phenyl group neither
and thus its insertion in the molecule do not justify. The contribution of the R’ group
in the potency, as indicated by GE values, is less prominent. In the case of chlorine
atom in 1e, negative GE value was observed, stating the negative contribution of this
atom to the affinity. The best contribution was given by the methoxy group of 1g (GE
= 0.35).
In summary, the dihydrobenzofuranyl-methylpiperazine (LINS01) molecules are
selective H3R antagonists with no intrinsic activity at either the human histamine
H1R, H2R or H3R and no, or extremely poor, affinity for the H1R or H2R. Although the
best ligands (1g and 1h) have greater than 100-fold H3R selectivity (over H1R or
H2R), their affinities for the human H3R (66 nM and 100 nM) clearly show potential
scope for further improvement with different chemical modifications. As future
directions, this chemical scaffold should be used to design novel derivatives in order
to increase the affinity to H3R taking advantage on the selectivity profile of such
compounds.
Conflicts of interest
The authors declare none.
Acknowledgements
The authors would like to thank CNPq (grant no. 455411/2014-0) and FAPESP
(2016/25028-3) for providing financial support and fellowship to M.F.C. (2016/23139-
2) and to G.A.B.F. (2017/05441-6) and Richard Proudman for technical assistance
with cell culture.
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Figure Captions
Figure 1. Structure of LINS01 compounds.
Figure 2. Reagents and conditions. a. Phenol, allyl bromide (2 eq.), K2CO3 (2 eq.),
acetone, 60 ºC, ))) 4h or conventional 12 h; b. 200 ºC, MW (300 W max., 300 psi
max.), DMF, 1 h; c. I2 (1.1 eq.), water, 4-8 h; d. Piperazines (NH, N-Me, N-allyl, N-
Ph, 2-4 eq.), K2CO3 (1 eq.), THF, 80 ºC, 24 h or 1-phenylpiperazine (1.1 eq.), MW
(300 psi max, 300 W max), 120 ºC, 1.5 h.
Figure 3. Reporter gene production in response to in the absence and presence of
different antagonist ligands in CHO-H1-SRE-luciferase cells (a and b), CHO-H2-
CRE-SPAP cells (c and d) and CHO-H3-CRE-SPAP cells (e and f). Bars represent
basal CRE-SPAP production, that in response to 10 µM histamine, 10 µM PDBU, 3
µM forskolin, or antagonist alone. Data points are mean ± SEM of triplicate
determinations. These single experiments are representative of 4 (a), 8 (b), 4 (c), 8
(d), 4 (e) and 8 (f) separate experiments.
Figure 4. Reporter gene production in response in response to different LINS01
ligands CHO-H1-SRE-luciferase cells (a and b), CHO-H2-CRE-SPAP cells (c and d)
and CHO-H3-CRE-SPAP cells (e and f). Bars represent basal CRE-SPAP
production, that in response to 10 µM histamine, 10 µM PDBU or 3 µM forskolin
alone. Data points are mean ± SEM of triplicate determinations. These single
experiments are representative of 4 separate experiments in each case.
Figure 5. Proposed interactions of H3R ligands with Glu206 (blue dashed lines). (A)
UCL2190; (B) DL-80; (C) LINS01009 (1g); (D) Alignment of UCL2190 (yellow), DL-
80 (purple) and LINS01009 (grey) considering the aromatic ring and basic nitrogen
involved in the interaction with Glu206.
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Tables
Table 1. pKD values for the human H1R, H2R and H3R.
Compounds
H1R pKD (n)
5.27 ± 0.10 (4)
5.66 ± 0.10 (4)
<5.00 (4)
H2R pKD (n)
<5.00 (4)
<5.00 (4)
<5.00 (4)
<5.00 (4)
<5.00 (8)
<5.00 (4)
<5.00 (10)
<5.00 (8)
<5.00 (8)
<5.00 (8)
8.71 ± 0.04 (9)
<5.00 (8)
<5.00 (4)
H3R pKD (n)
1a
<5.00 (4)
1b
6.67 ± 0.07 (5)
6.87 ± 0.07 (5)
5.61 ± 0.13 (4)
6.31 ± 0.06 (8)
6.80 ± 0.10 (6)
7.18 ± 0.04 (15)
6.99 ± 0.02 (8)
6.29 ± 0.03 (8)
<5.00 (9)
1c
1d
<5.00 (4)
1e
<5.00 (8)
1f
<5.00 (4)
1g
<5.00 (10)
<5.00 (8)
1h
1i
5.87 ± 0.04 (8)
8.53 ± 0.05 (9)
<5.00 (9)
mepyramine
ICI162846
clobenpropit
JNJ-7777120
<5.00 (9)
5.01 ± 0.11 (9)
<5.00 (4)
9.18 ± 0.06 (9)
5.45 ± 0.07 (4)
pKD values for ligands as determined in reporter assays from the rightward parallel
shift of a histamine concentration response curve. Values are mean ± SEM of n
separate determinations.
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Table 2. Ligand metric analyses of the LINS01 compounds considering the H3R
affinity.
Compounds
clogP
1.30
1.90
2.54
3.59
2.73
2.32
1.93
3.58
4.02
LE
LELP
<3.04
3.53
LLE
FQ
GE
1a
1b
1c
1d
1e
1f
>0.43
0.54
0.50
0.35
0.48
0.52
0.52
0.46
0.37
0.62
>3.70
4.77
4.33
2.02
3.58
4.48
5.25
3.41
2.27
6.51
>0.56
0.74
0.74
0.59
0.69
0.74
0.78
0.74
0.65
0.98
-
>2.29a
>0.85a
>0.14a
-0.49b
0.18b
0.35b
0.11b
0.93c
-
5.13
10.28
5.68
4.48
1g
1h
1i
3.73
7.85
10.73
4.25
clobenpropit 2.67
aCalculated for the R-group based on 1a.
bCalculated for the R’-group based on 1b.
cCalculated for R’-group based on 1f.
This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.